Simultaneous DNA Methylation and Mutation Detection
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Solution Overview
Problem
Current methods for simultaneously detecting DNA methylation and mutations in a single assay are inefficient due to the need for bisulfite treatment, which can degrade DNA and require splitting samples, making them time-consuming and less effective for clinical samples.
Innovation Solution
A method that uses a modifying reagent to distinguish between methylated and non-methylated cytosine residues, allowing for simultaneous mutation and methylation detection in the same sample through nucleic acid amplification and specific primer hybridization, eliminating the need for sample splitting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used to detect DNA methylation, then methylation status can be determined, but DNA degradation occurs and sample splitting is required
Solution Approach 1:
The patent divides the detection process into two separate detection pathways: one for methylation status (using bisulfite treatment) and one for mutation status (using untreated DNA). By processing different portions of the sample through different treatment protocols, the method enables simultaneous detection of both methylation and mutation markers without compromising DNA integrity in either pathway.
Solution Approach 2:
The invention extracts and detects specific methylation markers from the DNA sample through bisulfite treatment, separating the methylation detection function from the mutation detection function. This allows the methylation analysis to be performed on a treated portion while the mutation analysis is performed on an untreated portion, eliminating the need to choose between the two detection methods.
2Measurement precision
If bisulfite treatment is applied to detect methylation, then methylation status is revealed, but the process becomes time-consuming and reduces productivity
Solution Approach 1:
The patent performs preliminary separation of the DNA sample into multiple portions before treatment. By pre-allocating different sample portions for different detection purposes (methylation vs. mutation), the method eliminates the need for time-consuming sample splitting after bisulfite treatment, thereby improving overall detection efficiency and productivity.
3Adaptability or versatility
If sample splitting is performed for simultaneous mutation and methylation detection, then both markers can be assessed, but diagnostic efficiency decreases
Solution Approach 1:
The method segments the DNA sample into multiple aliquots at the beginning of the process, with each aliquot designated for specific detection purposes. This upfront segmentation enables parallel processing of methylation and mutation analysis without requiring intermediate sample splitting, thereby maintaining diagnostic efficiency while achieving dual detection capability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid and reliable detection of methylation and mutation status in a single sample, improving diagnostic efficiency and reducing sample degradation, thus enhancing predictive profiling for disease and treatment outcomes.
Implementation Method 1
Bisulfite attaches itself to the C-6 of the cytosine ring. Subsequently, under alkaline conditions, the sulfonated cytosine is deaminated and desulfonated to uracil.
Implementation Method 2
Bisulfite attaches itself to the C-6 of the cytosine ring. Subsequently, under alkaline conditions, the sulfonated cytosine is deaminated and desulfonated to uracil.
Implementation Method 3
amplification is performed using a primer that hybridizes specifically to the mutated target sequence and a primer that hybridizes specifically to methylated DNA
Implementation Method 4
nucleic acid amplification processes to distinguish between mutated and non-mutated target sequence
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3B
AI summary
Disclosed are methods for assessing the rnethylation and mutation status of nucleic acid in a sample. The methods provide for methylation-dependent modification of the nucleic acid in a sample, and subsequently nucleic acid amplification processes to distinguish between mutated and non-mutated target sequence.